As described in detail in our progress report, the data generated over the past three years of funding demonstrate there is a network of heterochronic gene products, including let-7 microRNAs, IMP1, Hmga2, P16lnk4a and possibly p19Arf, that regulates temporal changes in stem cell function throughout life, including stem cell aging. During the next five-year funding period we propose to expand our understanding of this network by exploring new mechanisms that regulate the increase in P16lnk4a/p19Arf expression during aging. P16lnk4a is a cyclin-dependent kinase inhibitor and the Alternative Reading Frame (Arf) at the same locus encodes the p19Arf tumor suppressor. P16lnk4a/p19Arf expression is known to increase with age in mice and humans and is considered a biomarker of aging. The increase in P16lnk4a expression contributes to the decline in stem/progenitor cell function in multiple aging tissues and we are currently testing whether the increase in p19Arf expression also decreases neural stem/progenitor cell function and the regenerative capacity of aging tissues. Mechanisms that regulate P16lnk4a/p19Arf expression during aging are of interest to expand our understanding of the heterochronic gene network and its role in stem cell aging. We have shown previously that the polycomb complex 1 component, Bmi-1, is required in the early postnatal period to maintain forebrain stem cell function by negatively regulating P16lnk4a/p19Arf expression. Similar mechanisms promote stem cell maintenance in other tissues. However, a major limitation has been the inability to test whether Bmi-1 regulates adult stem cell function or aging. All of the studies published so far on Bmi-1 function in stem cells have been performed with germline knockout mice that die by two months of age. To test whether Bmi-1 regulates adult stem cell function, stem cell aging, and the increase in P16lnk4a/p19Arf expression during aging we have generated mice bearing a floxed allele of Bmi-i. In unpublished preliminary studies we find that conditional deletion of Bmi-1 from adult neural stem/progenitor cells leads to premature declines in forebrain stem/progenitor cell function and neurogenesis as well as a profound age-related increase in P16lnk4a/p19Arf expression. The magnitude of the increase in P16lnk4a/p19Arf expression climbed sharply with age, suggesting that Bmi-1 delays stem cell aging by opposing the increase in P16lnk4a/p19Arf expression. Our data also revealed requirements for Bmi-1 to prevent age-related behavioral deficits in motor function and balance that are associated with defects in proprioception and the premature development of cataracts. We propose to test whether Bmi-1 is required to prevent premature stem cell aging and the development of neurological deficits by opposing the age-related increase in P16lnk4a/p19Arf expression.